Lecture 9 Genome Instability and Checkpoints (WIP)

Question 1

What is meant by genome instability

Answer 1

An elevated rate of genetic alterations

Question 2

Tumorigenesis is a multi-stage process T or F

Answer 2

T

Question 3

Give some examples of checkpoints throughout the cell cycle

Answer 3

Restriction point that governs entry into S phase from G1 intra-S-phase checkpoint that checks for DNA damage or stalked replication forks G2M phase checkpoint that checks that all DNA has been replicated and then the metaphase to anaphase transition in mitosis that checks whether all chromosomes are attached to the spindle

Question 4

Why is the intestinal epithelium used as a model for the changes in cancer cells over time

Answer 4

This is the best characterised and understood cancer in terms of the chromosomal abnormalities that correspond with its progression

Question 5

Outline the progression of intestinal epithelium towards tumorigenesis

Answer 5

Normal epithelium hyperplastic epithelium early/intermediate/late adenoma carcinoma metastasis

Question 6

Below is a diagram outlining the progression of intestinal epithelium during tumorigenesis. Fill in above the arrows with the specific chromosomal abnormality that causes each transition

Answer 6

Loss of APC DNA hypomethylation activation of KRas loss of Smad4 loss of p53

Question 7

Give examples of some of the chromosomal changes that occur in the progression of cells during tumorigenesis

Answer 7

Loss of tumour suppressor genes proto-oncogene to oncogene transitions and changes in epigenetic regulation of the genome

Question 8

What is meant by a driver mutation

Answer 8

A mutation that emerges early in cancer and occurs in a large majority of all cancers of a certain type

Question 9

There are over 15 different driver mutations in colorectal cancer give some examples of these

Answer 9

APC PIK3CA KRas TP53 FBXW7 β-catenin axin-2

Question 10

Why are driver mutations better therapeutic targets in the development of cancer treatments

Answer 10

Because mutations in these genes are extremely common and occur early so this will maximise the patients who could benefit and would mean that the cancer is stopped early in its progression

Question 11

What is significant about the various different mutations that occur at each point in tumorigenesis

Answer 11

Mutations at each stage in tumorigenesis are often components of the same pathway

Question 12

What hypothesis was proposed to explain how tumour cells manage to accumulate mutations

Answer 12

The mutator hypothesis was proposed to explain the accumulation of all the mutations in tumour cells. This is the idea that tumour cells acquire initial mutator gene mutations that then increases the rate of subsequent mutations

Question 13

What are the two type of instability seen in cancer cells and how do they differ

Answer 13

Chromosomal instability – chromosomal rearrangements and aberrations as well as loss of heterozygosity aneuploidy/polyploidy and gene amplifications. Microsatellite instability – point mutations and base substitutions as well as microdeletions or insertions giving rise to missense mutations

Question 14

Microsatellite and chromosomal instabilities are common in normal cells T or F

Answer 14

F – they are extremely rare occurring once in every 10million cell divisions

Question 15

Where is it that mutator genes are often found

Answer 15

Form part of the DNA repair systems

Question 16

What are the different methods of DNA repair

Answer 16

Base excision repair nucleotide excision repair homologous recombination and non-homologous end joining

Question 17

What mechanisms exist to prevent DNA replication and processing errors

Answer 17

Precursor control polymerase proof-reading and mismatch repair

Question 18

What is meant by integral dependency. How does this relate to the cell cycle

Answer 18

Integral dependency is where one component is reliant on the previous component. It suggests that in order for the cell cycle to progress each phase needs to be completed before the next one can begin

Question 19

Give an example of integral dependency

Answer 19

Bacteriophage assembly – whereby one component of virus assembly depends on the previous component. In other words the way in which the virus assembles is inherent to the structure of the virus

Question 20

What is the effect of RAD9 mutations in the growth of S. cerevisiae

Answer 20

Cells lacking RAD9 are healthy in the absence of extrinsic interference. Hence the growth curves of RAD9 and rad9 yeast cells are the same

Question 21

What were the results of experiments carried out with yeast harbouring temperature-sensitive cdc9 mutations

Answer 21

FACS analysis of cdc9 mutants at permissive temperature looks normal however FACS analysis of cdc9 mutants at a restrictive temperature indicates G2 arrest

Question 22

What is the role of cdc9 and how does this account for what is seen in the temperature-sensitive mutants

Answer 22

Cdc9 codes for a DNA ligase that is important in lagging strand synthesis where it is required to join Okazaki fragments during DNA replication. If you lack cdc9 there will be complete DNA synthesis but this DNA will be fragmented. Hence cdc9 mutants at restrictive temperatures arrest after S phase as all of the DNA is synthesised but is fragmented so that M phase cannot proceed

Question 23

What is meant by cell viability how does it differ from cell cycle arrest

Answer 23

Viability is the ability of cells to grow and make daughter cells. It isn’t related to cell cycle arrest as in the case of temperature-sensitive mutants changing the permissive temperature of a cdc mutant back to a permissive one will result in the cells coming out of arrest and therefore being viable

Question 24

Describe what these results obtained by Lee Hartwell show about the viability of cdc9/rad9 mutants

Answer 24

These graphs show that wild type yeast as well as cdc9 and rad9 mutants are all viable a hence have a near 100% viability. However the rad9/cdc9 combination mutants have drastically reduced viability. Interestingly the longer you leave the rad9/cdc9 cells the more rapidly that viability is reduced. This implies that cells undergoing replication stress such as due to non-functional DNA ligase/cdc9 require RAD9 in order to maintain their viability. Once this is also lost cells are no longer viable

Question 25

How can loss of viability be used as a screen for checkpoint mutants

Answer 25

You can identify which mutations in cell cycle genes cause a loss of viability when crossed with rad9 mutants

Question 26

What kind of gene was RAD9 identified to be

Answer 26

A checkpoint gene

Question 27

What is meant by a checkpoint gene

Answer 27

An extrinsic control gene that ensures that viability can be maintained when an extrinsic stress is applied

Question 28

What is meant by mitotic catastrophe

Answer 28

Where cells attempt mitosis when they shouldn’t

Question 29

What is premature chromosome condensation

Answer 29

A phenotype associated with a loss of the S/M phase checkpoint. This is indicative of mitotic catastrophe where cells have attempted to enter mitosis without completion of S phase

Question 30

How has premature chromosome condensation lead to the identification of a checkpoint gene involved in the S phase to mitosis transition

Answer 30

BHK cells were mutagenized so as to harbour temperature-sensitive cell cycle mutations. These cells were the placed at the restrictive temperature before being treated with a drug used to stop cells in mitosis so as to look at the chromosome condensation. This lead to the identification of premature chromosome condensation indicative of a checkpoint gene

Question 31

What is the drug used to arrest cells in M phase

Answer 31

Colchicine

Question 32

Small molecular weight chemicals can have the same effects as RAD9 mutations in yeast and cause premature chromosomal condensation T or F

Answer 32

T - mitosis and the completion of DNA replication can be uncoupled by treating cells with caffeine. This leads to premature chromosome condensation and DNA fragmentation due to a premature entry into mitosis